Intracranial self-stimulation-reward or immobilization-aversion had different effects on neurite extension and the ERK pathway in neurotransmitter-sensitive mutant PC12 cells.

Various actions trigger pleasure (reward) or aversion (punishment) as emotional responses. Emotional factors that negatively affect brain neural control processes for long periods of time might cause various mental diseases by inducing neuronal changes. In the present study, newly developed PC12m12 cells which are　highly sensitivity to neurotransmitters such as acetylcholine (ACh), were used. Exposing the cells to plasma from rats that had been subjected to intracranial self-stimulation (ICSS) markedly upregulated neurite outgrowth. In addition, voluntary running in a wheel or forced on a rotating rod was used to induce behavioral excitation in rats, and examinations of their plasma confirmed that the ICSS-induced neurite outgrowth was not associated with the ICSS behavior itself. Furthermore, immunoblotting and treatment with U0126, an ERK (extracellular signal-regulated kinase) antagonist, showed that the ICSS-induced neurite outgrowth was related to neuronal ERK activity. Exposing the same cells to plasma from rats that had been subjected to immobilization (IMM) also increased neurite outgrowth. Although the degree of enhancement was not as great as that seen after the ICSS rat plasma treatment, it was less than that observed after treatment with ACh as a positive control. These results indicate that ICSS or IMM lead to varying degrees of morphological changes, such as enhanced neurite outgrowth, in PC12m12 cells, but the neuronal signal transduction pathways underlying these effects differ; i.e.,the former morphological change might involve the activation of the ERK pathway, whereas the latter changes might not. Using PC12m12 cells which exhibit sensitivity to neurotransmitters, it might be possible to clarify the pathogeneses of mental diseases at the neuronal level and search for therapeutic drugs.

C-SH2 point mutation converts p85ß regulatory subunit of phosphoinositide 3-kinase to an anti-aging gene.

Insulin interacts with the insulin receptor, and the activated receptor promotes activity of the phosphoinositide-3 kinase (PI3K) enzyme. A decrease in insulin or insulin-like growth factor 1 (IGF-1) signaling increases the lifespan in mammalian species. We found that a point mutation in the C-SH2 domain of the p85ß regulatory subunit of PI3K results in a prolonged lifespan. In p85ß mutant cells, nerve growth factor (NGF) activates the longevity protein FOXO, and the mutant p85ß gene produces strong resistance to oxidative stress, which contributes to aging. The p85ß gene mutation causes increased serum insulin and low blood glucose in p85ß mutant transgenic mice. Our results indicate that the p85ß mutant allele alters the activity of downstream targets of PI3K by NGF and platelet-derived growth factor (PDGF) but not by insulin. We report that a point mutation in the C-SH2 domain of p85ß transforms p85ß into a novel anti-aging gene by abnormally regulating PI3K.

Inhibition of inflammatory-molecule synthesis in THP-1 cells stimulated with phorbol 12-myristate 13-acetate by brefelamide derivatives.

Plasma osteopontin (OPN) levels are elevated in tuberculosis patients and may involve granuloma formation. New inhibitors using brefelamide, an aromatic amide isolated from Dictyostelium cellular slime molds that may inhibit OPN transcription in A549 cells at 1 µM concentration, were synthesized as compounds C, D, and E. Their inhibitory activity against OPN synthesis in phorbol 12-myristate 13-acetate (PMA)-stimulated THP-1 cells was confirmed using enzyme-linked immunosorbent assay (ELISA), a multicolor immune-fluorescent microscope, and western blot. In the ELISA performed using full-length OPN, each compound showed significant inhibition in culture supernatants with half maximal inhibitory concentration (IC50) values of 1.6, 1.8, and 2.2 µM for C, D, and E, respectively. In another ELISA to detect the immune-related form of OPN, IC50 values were 0.6, 1.2, and 2.5 µM for compounds C, D, and E, respectively. The decreases in OPN expression and synthesis were confirmed using immunofluorescence and western blot studies using compound-treated cells or cell lysates. Luminex assay of the supernatants of PMA-treated THP-1 cells showed significant reduction in the synthesis of interleukin (IL)-1ß, galectin-9, and tumor necrosis factor (TNF)-α. Elucidation of the detailed mechanisms of the biological activities of these compounds would be necessary; however, they may be used in clinical trials for infectious diseases, inflammatory disorders, and cancer.

Synthesis of a Cleaved Form of Osteopontin by THP-1 Cells and Its Alteration by Phorbol 12-Myristate 13-Acetate and BCG Infection.

The protease-cleaved osteopontin (OPN) was proposed to enhance the migration of memory T cells to granulomas in tuberculosis. Various forms of OPN were identified in human monocytic THP-1 cells stimulated by phorbol 12-myristate 13-acetate (PMA). Antibodies O-17, 10A16 and 34E3, which recognize N-terminus, the C-half, and thrombin-cleaved site of OPN, respectively, all detected distinct bands on Western blots following PMA stimulation. Bands corresponding to 18 and 30 kD were detected by antibodies 34E3 and 10A16, indicating that OPN cleavage occurred by endogenous proteases in the PMA-stimulated THP-1 cells. In immune-fluorescence (IF) assay, 34E3 positive signals were detected in intracellular space of non-infected and bacillus Calmette-Guérin (BCG)-infected cells; however, 10A16 positive signals were confirmed in extracellular area in PMA-stimulated cells followed by BCG infection. Small amounts of full-length (FL) and thrombin-cleaved (Tr) OPN were detected by ELISA in the supernatants of non-PMA-stimulated cells, and increased levels of all forms, including undefined (Ud) OPN, in PMA-stimulated cells. ELISA showed a decrease in OPN synthesis during BCG infection. To our knowledge, this is the first report of OPN cleavage in THP-1 macrophages after PMA stimulation, and of enhanced cleavage induced by BCG infection.

Intracranial self-stimulation and immobilization had different effects on neurite extension and the p38 MAPK pathway in PC12m3 cells.

AIM: In mammals, rewarding and aversive states are motivational drivers of behavioral expression. However, it is unclear whether such states affect neuronal functions at the level of individual neurons. In the present study, the neuronal effects of rewarding and aversive states were investigated in using PC12 mutant cells (PC12m3 cells) with low sensitivity to nerve growth factor. MAIN METHODS: The intracranial self-stimulation (ICSS) and immobilization (IMM) methods were used to create rewarding and aversive states, respectively, in rats. Furthermore, experiments involving voluntary running on a wheel and forced running on a rotating rod were used to evaluate the effects of behavioral excitement on neurons. Then, the effects of plasma samples collected from the animals on neurite extension were examined microscopically, and p38 mitogen-activated protein kinase (MAPK) activity was assessed using Western blotting. KEY FINDINGS: Plasma samples from the ICSS and IMM rats facilitated neurite outgrowth to different degrees. However, their effects were not influenced by behavioral excitement. Furthermore, the plasma from the ICSS rats also induced upregulated p38 MAPK activity, whereas that from the IMM rats produced the same or slightly lower levels of MAPK activity to the control plasma. SIGNIFICANCE: These findings indicate that rewarding and aversive states might cause morphological changes, such as neurite extension. As for the effects of these states on p38 MAPK activity, the former state might directly increase p38 MAPK activity, but the latter state might have no effect or cause a slight reduction in p38 MAPK activity.

Intracranial self-stimulation-reward induces neurite extension in PC12m3 cells and activation of the p38 MAPK pathway.

Factors that trigger emotional expression may be divided into two patterns according to the type of motivation, acquiring reward (pleasure) and avoiding aversion (punishment). Repeated exposure to certain external stimuli accompanied by aberrant motivation may produce psychiatric diseases such as bipolar disorder and addiction via dysregulation of the central nervous system. However, neurobiological underpinnings of such diseases have not been clarified, especially at the neuronal level. In the present study, plasma from rats undergoing intracranial self-stimulation (ICSS) produced neurite outgrowth in PC12-variant cells (PC12m3). Stimulated PC12m3 cells also exhibited heightened activity of the p38 MAPK pathway. These findings indicate that reward states lead to not only morphological changes but also increases in p38 MAPK activity at the neuronal level in the central nervous system.

Neurite outgrowth of PC12 mutant cells induced by orange oil and d-limonene via the p38 MAPK pathway.

We studied the effects of natural essential oil on neurite outgrowth in PC12m3 neuronal cells to elucidate the mechanism underlying the action of the oils used in aromatherapy. Neurite outgrowth can be induced by nerve growth factor (NGF), where ERK and p38 MAPK among MAPK pathways play important roles in activating intracellular signal transduction. In this study, we investigated whether d-limonene, the major component of essential oils from oranges, can promote neurite outgrowth in PC12m3 cells, in which neurite outgrowth can be induced by various physical stimulations. We also examined by which pathways, the ERK, p38 MAPK or JNK pathway, d-limonene acts on PC12m3 cells. Our results showed that neurite outgrowth can be induced when the cells are treated with d-limonene. After treatment with d-limonene, we observed that p38 MAPK is strongly activated in PC12m3 cells, while ERK is weakly activated. In contrast, JNK shows little activity. A study using an inhibitor of p38 MAPK revealed that neurite outgrowth in PC12m3 cells is induced via the activation of p38 MAPK by d-limonene. The results thus indicate that d-limonene may promote neural cell differentiation mainly via activation of the p38 MAPK pathway.

Aluminum toxicity recovery processes in root apices. Possible association with oxidative stress.

Al inhibits root apex elongation with concomitant morphological injuries such as ruptures punctuated by the regions stained with Evans blue. The recovery can be investigated by transfer of Al-injured roots to a solution lacking Al. In the Al-injured root apex, superoxide anion, H(2)O(2), Al, and lignin accumulate. During the recovery process, the central cylinder elongates leaving the region stained with Evans blue without marked disappearance. The obvious function of the region is not clear but may trigger the elongation of central cylinder during the recovery process. Thus the function of the region stained with Evans blue might be derived from the programmed cell-like idea. Oxidative stress concerns events induced under Al toxicity and the recovery process. The superoxide anion is primarily formed by plasma membrane-associated NADPH oxidase and is dismuted to H(2)O(2) and O(2) by superoxide dismutase. H(2)O(2) provides the electrons for the polymerization of phenolics to lignin, which causes the stiffening of the cell wall. The distortion of the cell wall caused by lignin may induce the breaking and tearing of cells, which results in the formation of ruptures at the rhizodermis and outer cortex layers. The production of superoxide anion, H(2)O(2), and lignin was reduced during the recovery process and thereby the elongation of the central cylinder may be induced.

Changes in rupture formation and zonary region stained with Evans blue during the recovery process from aluminum toxicity in the pea root apex.

We investigated how the pea (Pisum sativum cv. Harunoka) root, upon return to an Al-free condition, recovers from injury caused by exposure to Al. Elongation and re-elongation of the root during the recovery process from Al injury occurred only in the apical 5-mm region of the pea root. With the model system of the pea root for recovery from Al injury, images of the root characterized by zonal staining with Evans blue showed the existence of two regions in the root apex consisting of rupture and zonary stained regions. Ruptures enlarged by increase in their depth but without widening of the intervals among zonary stained regions in the roots treated with Al continuously. On the other hand, intervals of the zonary stained regions were widened due to re-elongation of the root and were narrow in the rupture region in the recovery root.

Differential response of heat-shock-induced p38 MAPK and JNK activity in PC12 mutant and PC12 parental cells for differentiation and apoptosis.

Among the 3 mitogen-activated protein kinases--ERK, p38 MAPK and JNK--JNK has been suggested to participate in apoptosis, whereas p38 MAPK is thought to be part of the differentiation response. There are many common inducers of JNK and p38 MAPK, but the mechanisms underlying the differential response to apoptosis and differentiation are poorly understood. We found that heatshock activated p38 MAPK at 3 min after exposure to a temperature of 44 degrees C in stress-hypersensitive PC12m3 mutant cells, while it activated JNK at 20 min after the same heat treatment. However, heatshock activated p38 MAPK 5min after heat treatment and JNK 10 min after heat treatment in PC12 parental cells. The extent of phosphorylation of p38 MAPK induced by heat shock in PC12m3 cells was significantly greater than that in PC12 parental cells, and a high level of heat-shock-induced neurite outgrowth was observed only in PC12m3 cells. On the other hand, heat-shock-induced JNK activation appeared more quickly and apoptosis started earlier in PC12 parental cells. These findings indicate that short stress induces p38 MAPK and longer stress induces JNK, and that the response of these kinases to heat shock differs depending on cell type.

Heat shock-induced three-dimensional-like proliferation of normal human fibroblasts mediated by pressed silk.

The aim of this study was to determine the optimal heat treatment conditions for enhancement of pressed silk-mediated 3D-like proliferation of normal human dermal fibroblasts, as well as to determine the responses to heat shock of cells and intracellular signaling pathways. The beginning of 3D-like pattern formation of cells was observed in the second week after the start of the experiment. The mean rates of beginning of 3D-like pattern formation by cells heat-treated at 40 masculineC and 43 masculineC for 10 min were significantly higher (3.2- and 8.6-fold, respectively) than that of untreated cells. We found that apoptosis had occurred in 7.5% and 50.0% of the cells at one week after heat treatment for 10 min at 43 masculineC and 45 masculineC, respectively. Western blot analysis demonstrated that phosphorylation of p38 MAPK and that of Hsp27 were markedly increased by heat treatment at 43 masculineC for 10 min. The results of an experiment using a p38 MAPK inhibitor and Hsp27 inhibitor suggest that activation of p38 MAPK by heat shock is associated with 3D-like cell proliferation and that Hsp27 contributes to the inhibition of apoptosis. The results of this study should be useful for further studies aimed at elucidation of the physiologic mechanisms underlying thermotherapy.

Artepillin C derived from propolis induces neurite outgrowth in PC12m3 cells via ERK and p38 MAPK pathways.

We investigated whether artepillin C, a major component of Brazilian propolis, acts as a neurotrophic-like factor in rat PC12m3 cells, in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of PC12m3 cells were treated with artepillin C at a concentration of 20 microM, the frequency of neurite outgrowth induced by artepillin C was approximately 7-fold greater than that induced by NGF alone. Artepillin C induced-neurite outgrowth of PC12m3 cells was inhibited by the ERK inhibitor U0126 and by the p38 MAPK inhibitor SB203580. Although artepillin C-induced p38 MAPK activity was detected in PC12m3 cells, phosphorylation of ERK induced by artepillin C was not observed. On the other hand, artepillin C caused rapid activation of ERK and the time course of the activation was similar to that induced by NGF treatment in PC12 parental cells. However, NGF-induced neurite outgrowth was inhibited by artepillin C treatment. Interestingly, inhibition of ERK by U0126 completely prevented artepillin C-induced p38 MAPK phosphorylation of PC12m3 cells. These findings suggest that artepillin C-induced activation of p38 MAPK through the ERK signaling pathway is responsible for the neurite outgrowth of PC12m3 cells.

Microwave irradiation induces neurite outgrowth in PC12m3 cells via the p38 mitogen-activated protein kinase pathway.

The increasing use of mobile phone communication has raised concerns about possible health hazard effects of microwave irradiation. We investigated damage and differentiation caused by microwave irradiation on drug-hypersensitive PC12 cell line (PC12m3). These cells showed enhancement of neurite outgrowth to various stimulants. The frequency of neurite outgrowth induced by 2.45 GHz (200 W) of microwave irradiation was approximately 10-fold greater than that of non-irradiated control cells. Incubation of PC12m3 cells with SB203580, a specific inhibitor of p38 MAPK, resulted in marked inhibition of the microwave radiation-induced neurite outgrowth. Also, activation of the transcription factor CREB induced by microwave irradiation was inhibited by SB203580. Heat shock treatment at 45 degrees C had a strong toxic effect on PC12m3 cells, whereas microwave treatment had no toxic effect on PC12m3 cells. These findings indicate that p38 MAPK is responsible for the survival of PC12m3 cells and might induce neurite outgrowth via a CREB signaling pathway when subjected to microwave irradiation.

The Membrane Topology of ALMT1, an Aluminum-Activated Malate Transport Protein in Wheat (Triticum aestivum).

The wheat ALMT1 gene encodes an aluminum (Al)-activated malate transport protein which confers Al-resistance. We investigated the membrane topology of this plasma-membrane localized protein with immunocytochemical techniques. Several green fluorescent protein (GFP)-fused and histidine (His)-tagged chimeras of ALMT1 were prepared based on a computer-predicted secondary structure and transiently expressed in cultured mammalian cells. Antibodies raised to polypeptide epitopes of ALMT1 were used in conjunction with the antibody to the His-tags to determine the topology of ALMT1. This study shows that the ALMT1 protein contains six transmembrane domains with the amino and carboxyl termini located on the extracellular side of the plasma membrane.

Mutations in two matrix metalloproteinase genes, MMP-2 and MT1-MMP, are synthetic lethal in mice.

The matrix metalloproteinase (MMP) family (approximately 25 members in mammals) has been implicated in extracellular matrix remodeling associated with embryonic development, cancer formation and progression, and various other physiological and pathological events. Inactivating mutations in individual matrix metalloproteinase genes in mice described so far, however, are nonlethal at least up to the first few weeks after birth, suggesting functional redundancy among MMP family members. Here, we report that mice lacking two MMPs, MMP-2 (nonmembrane type) and MT1-MMP (membrane type), die immediately after birth with respiratory failure, abnormal blood vessels, and immature muscle fibers reminiscent of central core disease. In the absence of MMP-2 and MT1-MMP, myoblast fusion in vitro is also significantly retarded. These findings suggest functional overlap in mice between the two MMPs with distinct molecular natures.

Mechanism of gene expression of Arabidopsis glutathione S-transferase, AtGST1, and AtGST11 in response to aluminum stress.

The gene expression of two Al-induced Arabidopsis glutathione S-transferase genes, AtGST1 and AtGST11, was analyzed to investigate the mechanism underlying the response to Al stress. An approximately 1-kb DNA fragment of the 5'-upstream region of each gene was fused to a beta-glucuronidase (GUS) reporter gene (pAtGST1::GUS and pAtGST11::GUS) and introduced into Arabidopsis ecotype Landsberg erecta. The constructed transgenic lines showed a time-dependent gene expression to a different degree in the root and/or leaf by Al stress. The pAtGST1::GUS gene was induced after a short Al treatment (maximum expression after a 2-h exposure), while the pAtGST11::GUS gene was induced by a longer Al treatment (approximately 8 h for maximum expression). Since the gene expression was observed in the leaf when only the root was exposed to Al stress, a signaling system between the root and shoot was suggested in Al stress. A GUS staining experiment using an adult transgenic line carrying the pAtGST11::GUS gene supported this suggestion. Furthermore, Al treatment simultaneously with various Ca depleted conditions in root region enhanced the gene expression of the pAtGST11::GUS in the shoot region. This result suggested that the degree of Al toxicity in the root reflects the gene response of pAtGST11::GUS in the shoot via the deduced signaling system. Both transgenic lines also showed an increase of GUS activity after cold stress, heat stress, metal toxicity, and oxidative damages, suggesting a common induction mechanism in response to the tested stresses including Al stress.

Immunosuppressant FK506 induces neurite outgrowth in PC12 mutant cells with impaired NGF-promoted neuritogenesis via a novel MAP kinase signaling pathway.

We obtained a drug-hypersensitive PC12 mutant cell (PC12m3), in which neurite outgrowth was strongly stimulated by various drugs such as FK506, calcimycin and cAMP, under the condition of NGF treatment. The frequency of neurite outgrowth stimulated by FK506 was approximately 40 times greater than by NGF alone. The effects of FK506 on neurite outgrowth in PC12m3 cells were inhibited by rapamycin, an FK506 antagonist, and by calcimycin, a calcium ionophore. PC12m3 cells had a strong NGF-induced MAP kinase activity, the same as PC12 parental cells. However, FK506-induced MAP kinase activity was detected only in PC12m3 cells. The activation of MAP kinase by FK506 in PC12m3 cells was markedly inhibited by rapamicin and calcimycin. FK506-induced MAP kinase activity was also inhibited by MAP kinase inhibitor U0126. These results demonstrate that drug-hypersensitive PC12m3 cells have a novel FK506-induced MAP kinase pathway for neuritogenesis.